Lab Report 2 food microbiology

Introduction Aerobic plate count (APC), also known as standard plate count or the total viable count is one of the recommended tests applied to identify the presence of microbes in the food. Some food industry applies APC to ensure the quality of food is suitable for human consumption. APC measures only that microbial cell able to grow to visible and separate colonies. Standardized test condition is provided for APC which is the sample used must incubated for a sufficient period of time in an aerobic atmosphere and at a suitable temperature. Besides, the sample microbial cell for APC is present in an analytical unit which the colonies are mixed with agar containing appropriate nutrients. This analytical unit can be referred as colony forming units (CFU). In fact, CFU is more preferable by Microbiologist or Scientist instead of total bacteria counts in a sample. This is because the dead bacteria and the bacteria which cannot replicate under conditions being tested can be ignore. Futhermore, a new method, Petrifilm Aerobic Count is also used by food industry nowsaday. Petrifilm plates are thin film, sample-ready, dehydtated versions of conventional Petri agar plate. To improve its visibility, there is a special medium on the film. The medium is containing a colourless tetrazolium salt which bacteria use as an electron acceptor reducing the compound to a coloured product, tetrazolium formazan. Hence, Petrifilm are ready to use immediately after taking out of their packet.

Objective

1. To learn how to prepare and dilute a food sample properly. 2. To practice how to pour agar and mix sample correctly and use the Petxrifilm correctly. 3. Be able to count colonies according to standard rules of counting. 4. To apply the standard rules of counting to various conditions.

Method Pour plate method 1.

All data from the product was recorded.

2.

11g of milk was diluted to consecutive dilution which are 10

−1

−2

, 10

and

10−3 . 3.

The dilution bottles with the appropriate dilution was obtained and labeled.

4.

Two Petri plates for each assigned dilution were obtained and each of the

duplicate plates was marked with appropriate dilution. 5.

Sterile weighing paper, or aluminum foil, or use the dilution blank or blender

directly was obtained. The weight was tarred on the balance. 6.

The indicated amount of the assigned sample was weighed aseptically and

placed on the balance or in the blender or dilution blank with a flame spatula. 7.

A serial dilution was prepared according to the scheme. Pipets was get one at a

time only when it was needed. Never put pipets on the bench top. It can only in the original container or in hand. The aseptic technique was applied. Each dilution blank was shaken. 8.

When the dilution series has been made, each dilution was agitated into each

of the appropriately labeled Petri dishes with the beginning at the highest dilution. The last drop was expelled with a pipet aid. If the excess is replaced in the appropriate blank and the pipet rinsed with the next lower dilution, only a single pipet is required if plating is begun at the highest dilution.

9.

12 to 15ml of plate count agar was cooled to 44°C-46°C and poured into each

plate within 15 minutes of making original dilution. 10.

The sample was mixed immediately and the agar medium by rotating each

plate on a flat surface first in one direction, then the other in a gentle. 11.

The agar was allowed to solidify and the Petri dishes was inverted and

incubated at 35°C for 48±2 hours. Petrifilm method 1. All data was recorded from the product. 2.

−1 −2 11g of milk was diluted to consecutive dilution which are 10 , 10

−3 and 10 .

3. The dilution bottles with the appropriate dilution was obtained and labeled. 4. Two Petrifilm plates for each assigned dilution were obtained and each of the duplicate plates was marked with the appropriate dilution. The plates were laid and the dilution blanks out on the bench top in a pattern corresponding to the scheme. 5. Sterile weighing paper was obtained or aluminum foils, or use the dilution blankor blender directly. 6. The indicated amount of the assigned sample was weighed aseptically, and placed on the balance or in the blender or dilution blank with a flamed spatula. a. The sample was blended under a hood to avoid airborne contamination, or b. The dilution blank was shaking if added directly. 7. Serial dilutions were prepared according to the scheme. Pipets was get one at a time only when it was needed. Never put pipets on the bench top. It can only in the original container or in hand. The aseptic technique was applied. Each dilution blank was shaken. 8. When the dilution series has been made, each dilution was agitated into each of the appropriately labeled Petri dishes with the beginning at the highest dilution. The last drop was expelled with a pipet aid. If the excess is replaced in the appropriate blank and the pipet rinsed with the next lower dilution, only a single pipet is required if plating is begun at the highest dilution.

9. 12 to 15 ml of plate count agar (PCA) was cooled to 44°C-46°C and poured into each plate within 15 minutes of making the original dilution. 10. The sample and the agar medium were mixed immediately by rotating each plate on a flat surface first in one direction, then the other in a gentle. 11. The agar was allowed to solidify, inverted the Petri dishes and incubated it at 35°C for 48±2 hours (or 5 days at room temperature).

Results and Observations: (i) Pour plate method

Figure 1: Aerobic pour plates with dilution factor of 10-4.

Figure 2: Aerobic pour plates with dilution factor of 10-5.

Figure 3: Aerobic pour plates with dilution factor of 10-6.

Table 1.0: Aerobic pour plates with different dilution factors Dilution

Petri Dish 1

Petri Dish 2

Average count

Count

per

Factor 10-4

412

520

466

volume / weight TNTC

10-5

111

101

106

1.06 x 107 cfu/ml

10-6

9

13

11

TFTC

unit

Based on the table 1.0, the colonies obtained from the dilution factor of 10-4 and 10-6 are 466 and 11 respectively, which are out from the range of 25-250 colonies. The dilution factor of 10-5 contains 106 colonies, which is within the range of 25-250 colonies. Count =

106 10-5 x 1.0

= 1.06 x 107 cfu/ml

(ii) RIDAfilm method

Figure 5: Aerobic RIDAfilms from left are dilution factors of 10-4, 10-5, 10-6.

Table 2.0: Aerobic RIDAfilms with different dilution factors Food

Dilution factor

Average count

Count

10 10-5

1912 198

volume / weight TNTC 1.98 x 107 cfu/ml

10-6

28

2.8 x 107 cfu/ml

-4

Milk

per

unit

From table 2.0, only 10-4 dilution yields 1912 colonies which are out the range of 25 to 250 colonies. 10-5 and 10-6 dilutions yield 198 and 28 colonies respectively. Count =

198 10-5 x 1.0

= 1.98 x 107 cfu/ml Count =

28 10-6 x 1.0

= 2.8 x 107 cfu/ml Discussion: Pour plate method was a method used to detect the presence of viable bacterium and amplify itself to form visible colonies. In pour plate method, sample

(usually 1ml) was pipetted into the petri dish and mixed with the appropriate molten agar (Adams 2008). Pour plate method allowed the growth of spoil milk bacteria which was facultative anaerobe. Based on the experiment, table 1.0 showed that the bacteria from the spoil milk were higher in aerobic condition. This was because in the absence of anoxic jar, anaerobes bacteria were killed when exposed to oxygen as it must strictly grow under the absence of oxygen. Thus, pour plate method favoured the growth of aerobes bacteria. However, some of the obligate aerobes might grow poorly if it was deeply embedded in the agar (David 2015). From the results, there was 10600000 variable cells/ml in the dilution factor of 10-5, while the colonies obtained from the dilution factor of 10-4 and 10-6 were 466 and 11 respectively, which were out from the range of 25-250 colonies. This was because in the dilution factor of 10 -5 contained more microorganism, this was a condition of overcrowded happening. So, less visible colonies could be grown on the agar plate due to the competition among each other (Academia.edu 2016). To overcome the overcrowded problem, a ten-fold serial dilution was needed. However, we still could observe that there were colonies in the dilution factor of 10-4 and 10-6, but both were not suitable to be used to calculate the bacteria counts. This was because the concentration of bacteria which was within the range of 25-250 colonies only statistically suitable. Since the warm molten agar was poured onto the bacterial suspension, its temperature could damage or kill the heat-sensitive bacteria. As a result, no visible colonies could be found. For the aerobes bacteria also would be trapped inside the agar, leading to fail to survive as no oxygen could diffuse into the agar. Hence, pour plate method was considered as a selective technique. To avoid the damaging or killing of heat-sensitive bacteria, the agar should be leaved to become cooler but still in molten state before pouring onto the bacterial suspension (Academia.edu 2016). RIDAfilm method was a convenience, simple and effective method used to detect the presence of bacteria compare with other plating method such as pour plate method (Martin 2012). A fluid food product or a dilution of a solid food was placed to the dry culture medium overlaid with polyethylene film coated with water soluble gelling agent. RIDAfilm method was not statistically different from pour plate method for the enumeration of organisms in spoil milk. Based on the experiment, the colonies

count for dilution factor of 10-5 by using RIDAfilm method was 1.98 x 107 cfu/ml, which is higher than the count using pour plate method which yielded 1.06 x 10 7 cfu/ml. The difference of the value might due to experimental error. Thermal shock to the psychrotrophs might occur when the agar was poured into the petri dish in a hot condition. Thus, the colonies counts were affected. RIDAfilm method was easier to identify the presence of bacteria as red dye is used and the available of grid make counting of bacteria easier. For pour plate method, if the colonies grow was too crowded and was likely to result in many colony-forming colonies which eventually lead to underestimate of counting the presence of the bacteria (Martin 2012). Questions 1. Pipetting: a. Possibility of an error in my reading the volume accurately or in the volume itself. b. Tip damaged or broken c. No sterile. 2. Sample representation a. Amount of the sample does not large enough 3. Sample weighing a. Doesn’t blank the weighing scale when putting on the sterile weighing paper before adding the sample. 4. Dilution bottle a. Human error in calculation of dilution factors. b. Inaccurate pipetting. 5. Counting plates a. Miscounting colonies. b. Distinguish wrongly between food particles and bacteria

Conclusion Throughout the experiments, when working with media and reagents used to culture microorganisms, aseptic technique must be practiced to ensure contamination is minimized. To getting isolated colonies when you inoculate, serial dilution is needed. Beside, aerobic plate count relies on bacteria growing a colony on a nutrient medium so that the colony becomes visible to the naked eye

and the number of colonies on a plate can be counted. We must follow the standard rules when counting the colonies to avoid sampling error. APN give a good prediction to how long the food will take to spoil.

References: Academia.edu., 2016. LAB REPORT OF MICROBIOLOGY [Online]. Available from: http://www.academia.edu/8826259/LAB_REPORT_OF_MICROBIOLOGY [Accessed 6 June 2016]. Adams. M., 2008. Food Microbiology, 3rd ed. David,

B.F.,

2015.

Pour

Plate

Technique

[Online].

Available

from:

http://biology.clc.uc.edu/fankhauser/Labs/Microbiology/Meat_Milk/Pour_Pla e.htm [Accessed 6 June 2016]. Martin, A., 2012. Fisheries Processing: Biotechnological applications.